The objective of the propposed experiments is to obtain information on the cellular and molecular aspects of bone development and biological calcification in the embryonic chick limb. Our studies over the past seven years of support have led us to a detailed working hypothesis concerning the events which lead to the formation of long bones in the developing chick embryo. The focus of the experimentation is on questions related to cellular lineages of osteoblasts, influences over the differentiation pattern of osteogenic cells, the cellular lineage of osteoclasts and the interaction between various osteogenic phenotypes. Four separate projects are detailed which center their attention on two important experimental objectives: First, the isolation of populations of cells enriched for particular osteogenic phenotypes; and second, the identification of unique markers for each osteogenic phenotype. Project I attempts to isolate osteoblasts from tissues undergoing first bone formation in the developing limb and from limb mesenchymal cell cultures optimized for the expression of osteogenic characteristics. Project II attempts to isolate distinct phenotypes from remodeling cartilage. Project III attempts to analyze cells associated with the osteoclast lineage including frank osteoclasts from Day 19 chick embryo long bones and osteoclasts formed from their usual progenitor cells, the circulating monocytes. Project IV attempts to identify unique markers for each osteogenic phenotype by using monoclonal antibody technology in an attempt to isolate cell surface antigens specific for these individual phenotypes. A enrichment program will be used involving isolation of purified phenotypes with the identification of unique cell surface markers and then the repurification of these phenotypes using the markers and information obtained in the study of these individual phenotypes. Such a "circle of refinement" will enable us to obtain pure populations of individual osteogenic phenotypes as well as distrinctive phenotype-specific markers. The long range utility of having these developmental and phenotype-specific probes would be to address questions which related to the sequence of events involved in normal bone development, bone repair and eventually bone remodeling.